Molecular Basis of Reduced Potency of Underacylated Endotoxins

Abstract

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.

MD-2–Dependent Pulmonary Immune Responses to Inhaled Lipooligosaccharides

“…The differences in potency we observed between LOS wt and LOS msbB in inducing airway inflammatory responses in vivo were strikingly similar to differences previously observed in LBP-dependent in vitro activation by these LOS species of cells expressing mCD14, MD-2, and TLR4 (17). Our findings thus strongly suggest that the molecular requirements for airway responsiveness to endotoxin correspond closely to the require- Figure 4.…”

“…MD-2-null mice do not mobilize airway inflammatory responses to nasally instilled LOS aggregates but are responsive to purified monomeric LOS:MD-2 (unpublished observation), confirming that the loss of responsiveness of MD-2-null mice to endotoxin is due to the absence of MD-2. In vitro, differences in potency of hexaacylated versus underacylated endotoxin species in inducing TLR4-dependent cell activation reflect differences in the functional properties of endotoxin bound to MD-2 (17,19). Thus, the differences we observed in this study in the ability of LOS wt versus LOS msbB to induce airway inflammation most likely reflect differences in the functional properties of hexacylated versus pentacylated LOS bound to MD-2 in the airway.…”

“…Metabolically 14 C-labeled LOS was isolated from an acetate auxotroph of wild-type NMB and from a msbB mutant derived from NMB as previously described (5,6,17) (see the online supplement). Concentrations of LOS (endotoxin activity [units]; EU) were measured using the kinetic chromogenic Limulus Amebocyte Lysate assay as previously described (26) and indicated an activity of LOS wt of 18,000,000 EU/ml (1,200 mg/ml) and for LOS msbB 33,800,000 (2,250 mg/ml) (see the online supplement).…”

“…In general, the most potent endotoxin species are hexaacylated, whereas either less or more highly acylated endotoxins are substantially less potent inducers of host proinflammatory responses (15,18). Differences in the potency of hexa-, tetra-, and pentaacylated endotoxins reflect differences in the ability of endotoxin-bound MD-2 to induce activation of TLR4 (17,19). On the basis of these observations, it has been suggested that the elaboration of underacylated endotoxins by Pseudomonas aeruginosa early in the evolution of pulmonary infection in cystic fibrosis (15,20,21) and by Yersinia pestis in pneumonic plague (22) contributes greatly to the virulence of these airway bacterial pathogens by blunting early host inflammatory responses needed for efficient mobilization of host defenses.…”

“…Among the most important structural determinants of endotoxin activity are the number, chain length, and location of fatty acyl chains within lipid A (13)(14)(15)(16)(17). In general, the most potent endotoxin species are hexaacylated, whereas either less or more highly acylated endotoxins are substantially less potent inducers of host proinflammatory responses (15,18).…”